Heterogeneous nuclear ribonucleoprotein A3 is the liver nuclear protein binding to age related increase element RNA of the factor IX gene

Abstract

Background: In the ASE/AIE-mediated genetic mechanism for age-related gene regulation, a recently identified age-related homeostasis mechanism, two genetic elements, ASE (age-related stability element) and AIE (age-related increase element as a stem-loop forming RNA), play critical roles in producing specific age-related expression patterns of genes.

Principal finding: We successfully identified heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3) as a major mouse liver nuclear protein binding to the AIE-derived RNAs of human factor IX (hFIX) as well as mouse factor IX (mFIX) genes. HnRNP A3 bound to the AIE RNA was not phosphorylated at its Ser(359), while hnRNP A3 in the mouse liver nuclear extracts was a mixture of phosphorylated and unphosphorylated Ser(359). HepG2 cells engineered to express recombinant hFIX transduced with adenoviral vectors harboring an effective siRNA against hnRNP A3 resulted in a substantial reduction in hFIX expression only in the cells carrying a hFIX expression vector with AIE, but not in the cells carrying a hFIX expression vector without AIE. The nuclear hnRNP A3 protein level in the mouse liver gradually increased with age, while its mRNA level stayed age-stable.

Conclusions: We identified hnRNP A3 as a major liver nuclear protein binding to FIX-AIE RNA. This protein plays a critical role in age-related gene expression, likely through an as yet unidentified epigenetic mechanism. The present study assigned a novel functional role to hnRNP A3 in age-related regulation of gene expression, opening up a new avenue for studying age-related homeostasis and underlying molecular mechanisms.

Figure 1. EMSAs and SDS-PAGE analyses of ³²P-hFIX-AIE RNA/nuclear protein and ³²P-mFIX-AIE RNA/nuclear protein complexes.

A. EMSAs of ³²P-hFIX-AIE RNA with liver NEs. Various conditions tested are shown at the top. Brackets on the left indicate hFIX-AIE RNA probe-protein complexes and degraded ³²P-hFIX-AIE RNA as shown. The position of free ³²P-hFIX-AIE RNA is shown with a short horizontal bar on the left. Lanes 1, 2 and 5–10 contain ³²P-hFIX-AIE RNA. Lane 2, treated with RNase T1; lanes 3 and 4, null controls; lanes 5–7, with increasing amounts of liver NEs; lanes 8–10, with NEs (10 µg) and increasing amounts of cold hFIX-AIE RNA competitor. B. EMSAs of ³²P-mFIX-AIE or ³²P-hFIX-AIE RNA with liver NEs. Lanes 1–7 contains ³²P-mFIX-AIE RNA, while lanes 8 and 9 contain ³²P-hFIX-AIE RNA. Positions of the AIE RNA probe-protein complex, free AIE RNA probe and degraded probe are similarly shown as in A. Lane 1, without NEs; lane 2, treated with RNase T1; lanes 3 and 4, with increasing amounts of NEs; lanes 5 and 6, with NEs (10 µg) and increasing amounts of cold mFIX-AIE RNA competitor; lane 7, with NEs (10 µg) and cold hFIX-AIE RNA; lane 8, with NEs (10 µg); lane 9, with NEs (10 µg) and cold mFIX-AIE RNA. C. SDS-PAGE analysis of UV cross-linked ³²P-hFIX-AIE RNA/nuclear protein complex treated with RNase T1 (lane 1), RNase A/T1 (lane 2) and no RNase treatment (lane 3). Brackets a and b represent the positions of AIE RNA-protein complex without and with RNase-treatment, respectively. Size marker positions are shown on the left. D. SDS-PAGE analysis of UV cross-linked and RNase T1-treated ³²P-hFIX-AIE RNA incubated with increasing amounts of NEs. Positions for the AIE RNA/protein complex and degraded ³²P-hFIX-AIE RNA are shown with brackets on the right. Lane 1, protein size marker with sizes shown on the left. E. SDS-PAGE analysis after competitive EMSAs of ³²P-hFIX-AIE RNA with cold hFIX-AIE RNA (lanes 2–4), and with poly(U), poly(A) and poly(C) (lanes 5–6). Other conditions are similar to D.

Figure 2. 2DE analysis of UV cross-linked/RNase-treated ³²P-hFIX-AIE RNA/liver nuclear protein complex and its autoradiography.

A. Analytical scale 2DE analyses of the solution-phase IEF chamber solutions containing UV cross-linked/RNase-treated ³²P-hFIX-AIE RNA/protein complexes. Chamber solutions of pH 3–4.6, pH 4.6–5.4, pH 5.4–6.4 and pH 6.4–7.0 zones were subjected to analytical 2DEs using immobilized pH 4–7 gradient gels for IEF and 4–12% gradient gel SDS-PAGE, and the results are shown in panels a, b, c and d, respectively. B. Silver-stained 2DE gel of UV cross-linked and RNase-treated ³²P-hFIX-AIE RNA/nuclear protein complexes concentrated to pH 3–4.6 and pH 4.6–5.4 chambers in the solution phase IEF. Molecular size marker proteins are shown on the left edge of the gel with their sizes. Arrow indicates the protein spot position matching with the center of the major radioactive spot detected by autoradiography. C. Autoradiogram of the gel shown in B. Arrow indicates the center of the major radioactive spot, and the gel recovered from this center area was subjected to subsequent MALDI-TOF/MS analysis.

Figure 3. Amino acid sequences of mouse hnRNP A3 in complex with hFIX-AIE RNA and mFIX-AIE RNA.

Amino acid sequences of mouse liver hnRNP A3 (isoform hnRNP A3a) bound with hFIX-AIE RNA (A) and mFIX-AIE RNA (B), identified by MALDI-TOF/MS and subsequent Mascot search analyses, are shown in bald letters of its known complete sequence . Underlines indicate the sequence region absent in hnRNP A3b, a short isoform of hnRNP A3 generated by an alternative splicing . In the present mass-spectrometric analyses, no discrimination between hnRNP A3a and hnRNP A3b was made. Domain structures in analogy to other known hnRNP family proteins include RRM 1 (RNA recognition motif: aa 35–118), RRM 2 (RNA recognition motif: aa 126–205) and RGG motif (aa 211–379) –.

Figure 4. MALDI-TOF/MS spectra of peptides generated from mouse hnRNP A3 in complex with hnFIX-AIE RNA and mFIX-AIE RNA, and with no AIE RNA.

A. Cropped mass spectra of the 2DE protein spot containing the UV-irradiated and RNase-treated ³²P-hFIX-AIE RNA/hnRNP A3 complex. Mass-spectrum m/z 1910.8 corresponding to a tryptic peptide of hnRNP A3 (aa 356 through 377) with Ser³⁵⁹ unphosphorylated was reproducibly detected, while mass-spectrum m/z 1990.8 corresponding to the same peptide with phosphorylated Ser³⁵⁹ was not detected. B. Cropped mass spectra of the 2DE protein spot containing the UV-irradiated and RNase-treated ³²P-mFIX-AIE RNA/hnRNP A3 complex. Experimental conditions used and observations made were similar to those described in A. C. Cropped mass spectra of one of protein spots separated in 2DE of liver NEs (free of hnRNP-AIE RNA). Both spectra m/z 1910.9 and m/z 1990.8 were reproducibly detected.

Figure 5. Construction of siRNAs against hnRNP A3 and their effects on hnRNP A3 expression.

A. Suppressive effects of siRNA 1–6 (siRNA against hnRNP A3) on the hnRNP A3 mRNA level in 293T cells. Effects of siRNA were assessed in duplicates in 293T cells co-transfected with expression vector plasmids for hnRNP A3 and various siRNA expression vectors as shown in Table 1. Averages of two independent assays of each duplicated measurements per condition were normalized to siRNA 7 (scrambeled siRNA 6) for presentsation. Thin vertical lines with short horizontal bars represent ranges of the duplicated measurements. B. Relative hnRNP A3 mRNA levels in the mouse liver post-injection of adenoviral vectors harboring siRNA 7 and siRNA 6. Animals were injected with the adenoviral vectors harboring siRNA 7, siRNA 6 or PBS-saline (n = 4 for each condition), and on day 4 and day 14 post-injection, hnRNP mRNA levels in the liver were measured. The results are normalized to that of β-actin mRNA, and their mean relative values over that of the PBS-saline condition (no siRNA, and defined as 1) are shown. Thin vertical lines with short horizontal bars represent S.D.

Figure 6. Age-dependent levels of hnRNP A3 mRNA and protein in the mouse liver.

A. Age-related changes of the hnRNP A3 mRNA level determined by RPA. Total liver RNA preparation obtained from male C57BL6/J mice (n = 4 per age point) was subjected to quantification by RPA. The top and bottom panels are for hnRNP A3 and β-actin, respectively. Numbers at the bottom of the panels represent age of animals. Size marker positions are shown on the left. B. The age-related profile of the relative hnRNP A3 mRNA level normalized to that of β-actin shown in A. C. Western blot analysis of age-related hnRNP A3 protein levels in the mouse liver. Protein size marker positions are shown on the left, and age of animals are shown at the bottom. Two major protein bands corresponding to the known isoforms, hnRNP A3a and hnRNP A3b, are depicted with a and b with arrows on the right. D. The age-related increase profiles of hnRNP A3a and hnRNP A3b proteins observed in C.